Non-phosphorylated Neurofilament Research Articles

Neurochemical atlas of the rabbit spinal cord.

Complex neurophysiological and morphologic experiments require suitable animal models for investigation. The rabbit is one of the most successful models for studying spinal cord functions owing to its substantial size. However, achieving precise surgical access to specific spinal regions requires a thorough understanding of the spinal cord's cytoarchitectonic structure and its spatial relationship with the vertebrae. The comprehensive anatomo-neurochemical atlases of the spinal cord are invaluable for attaining such insight. While such atlases exist for some rodents and primates, none exist for rabbits. We have developed a spinal cord atlas for rabbits to bridge this gap. Utilizing various neurochemical markers-including antibodies to NeuN, calbindin 28kDa, parvalbumin, choline acetyltransferase, nitric oxide synthase, and non-phosphorylated heavy-chain neurofilaments (SMI-32 antibody)-we present the visualization of diverse spinal neuronal populations, various spinal cord metrics, stereotaxic maps of transverse slices for each spinal segment, and a spatial map detailing the intricate relationship between the spinal cord and the vertebrae across its entire length.

Vulnerability of neurofilament-expressing neurons in frontotemporal dementia

Frontotemporal dementia (FTD) is an umbrella term for several early onset dementias, that are caused by frontotemporal lobar degeneration (FTLD), which involves the atrophy of the frontal and temporal lobes of the brain. Neuron loss in the frontal and temporal lobes is a characteristic feature of FTLD, however the selective vulnerability of different neuronal populations in this group of diseases is not fully understood. Neurofilament-expressing neurons have been shown to be selectively vulnerable in other neurodegenerative diseases, including Alzheimer's disease and amyotrophic lateral sclerosis, therefore we sought to investigate whether this neuronal population is vulnerable in FTLD. We also examined whether neuronal sub-type vulnerability differed between FTLD with TDP-43 inclusions (FTLD-TDP) and FTLD with tau inclusions (FTLD-Tau). Post-mortem human tissue from the superior frontal gyrus (SFG) of FTLD-TDP (n = 15), FTLD-Tau (n = 8) and aged Control cases (n = 6) was immunolabelled using antibodies against non-phosphorylated neurofilaments (SMI32 antibody), calretinin and NeuN, to explore neuronal cell loss. The presence of non-phosphorylated neurofilament immunolabelling in axons of the SFG white matter was also quantified as a measure of axon pathology, as axonal neurofilaments are normally phosphorylated. We demonstrate the selective loss of neurofilament-expressing neurons in both FTLD-TDP and FTLD-Tau cases compared to aged Controls. We also show that non-phosphorylated neurofilament axonal pathology in the SFG white matter was associated with increasing age, but not FTLD. This data suggests neurofilament-expressing neurons are vulnerable in both FTLD-TDP and FTLD-Tau.

Cytoarchitectonic gradients of laminar degeneration in behavioural variant frontotemporal dementia.

Behavioral variant frontotemporal dementia (bvFTD) is a clinical syndrome primarily caused by either tau (bvFTD-tau) or TDP-43 (bvFTD-TDP) proteinopathies. We previously found lower cortical layers and dorsolateral regions accumulate greater tau than TDP-43 pathology; however, patterns of laminar neurodegeneration across diverse cytoarchitecture in bvFTD is understudied. We hypothesized that bvFTD-tau and bvFTD-TDP have distinct laminar distributions of pyramidal neurodegeneration along cortical gradients, a topologic order of cytoarchitectonic subregions based on increasing pyramidal density and laminar differentiation. Here, we tested this hypothesis in a frontal cortical gradient consisting of five cytoarchitectonic types (i.e., periallocortex, agranular mesocortex, dysgranular mesocortex, eulaminate-I isocortex, eulaminate-II isocortex) spanning anterior cingulate, paracingulate, orbitofrontal, and mid-frontal gyri in bvFTD-tau (n=27), bvFTD-TDP (n=47), and healthy controls (HC; n=32). We immunostained all tissue for total neurons (NeuN; neuronal-nuclear protein) and pyramidal neurons (SMI32; non-phosphorylated neurofilament) and digitally quantified NeuN-immunoreactivity (ir) and SMI32-ir in supragranular II-III, infragranular V-VI, and all I-VI layers in each cytoarchitectonic type. We used linear mixed-effects models adjusted for demographic and biologic variables to compare SMI32-ir between groups and examine relationships with the cortical gradient, long-range pathways, and clinical symptoms. We found regional and laminar distributions of SMI32-ir expected for HC, validating our measures within the cortical gradient framework. While SMI32-ir loss was relatively uniform along the cortical gradient in bvFTD-TDP, SMI32-ir progressively decreased along the cortical gradient of bvFTD-tau and included greater SMI32-ir loss in supragranular eulaminate-II isocortex in bvFTD-tau versus bvFTD-TDP (p=0.039). Using a ratio of SMI32-ir to model known long-range connectivity between infragranular mesocortex and supragranular isocortex, we found a larger laminar ratio in bvFTD-tau versus bvFTD-TDP (p=0.019), suggesting select long-projecting pathways may contribute to isocortical-predominant degeneration in bvFTD-tau. In cytoarchitectonic types with the highest NeuN-ir, we found lower SMI32-ir in bvFTD-tau versus bvFTD-TDP (p=0.047), suggesting pyramidal neurodegeneration may occur earlier in bvFTD-tau. Lastly, we found that reduced SMI32-ir related to behavioral severity and frontal-mediated letter fluency, not temporal-mediated confrontation naming, demonstrating the clinical relevance and specificity of frontal pyramidal neurodegeneration to bvFTD-related symptoms. Our data suggest loss of neurofilament-rich pyramidal neurons is a clinically relevant feature of bvFTD that selectively worsens along a frontal cortical gradient in bvFTD-tau, not bvFTD-TDP. Therefore, tau-mediated degeneration may preferentially involve pyramidal-rich layers that connect more distant cytoarchitectonic types. Moreover, the hierarchical arrangement of cytoarchitecture along cortical gradients may be an important neuroanatomical framework for identifying which types of cells and pathways are differentially involved between proteinopathies.

A controversial question: Can morphometry and clinical history be enough to diagnose hippocampal dysplasia?

The presence of dysmorphic neurons with strong cytoplasmatic accumulation of heavy non-phosphorylated neurofilament is crucial for the diagnostics of focal cortical dysplasia type II (FCDII). While ILAE's classification describes neocortical dysplasias, some groups have reported patients with mesial t abnormal neurons in the hippocampus of mesial temporal lobe epilepsy. Here we report a patient with such abnormal neurons in the hippocampus and compared it with previous reports of hippocampal dysplasia. Finally, we discuss the need for diagnostic criteria of hippocampal dysplasia.

Transient expression of heavy-chain neurofilaments in the perigeniculate nucleus of cats.

The perigeniculate nucleus (PGN) is a visual part of the thalamic reticular nucleus modulating the information transfer between the lateral geniculate nucleus and the visual cortex. This study focused on the postnatal development of the PGN in cats, using the SMI-32 antibody, which recognizes non-phosphorylated heavy-chain neurofilaments responsible for neuronal structural maturation and is also used as a marker for motion processing, or Y, stream. We questioned whether transient neuronal populations exist in the PGN and can they possibly be related to the Y processing stream. We uncovered a transient, robust SMI-32 staining in the PGN of kittens aged 0-34days with the significant decline in the cellular density of labeled cells in older animals. According to the double-labeling, in all examined age groups, perigeniculate SMI-32-immunopositive cells are part of the main parvalbumin-positive population. The maximal cellular density of the double-stained cells appeared in animals aged 10-28days. We also revealed that the most significant growth of perigeniculate cells's soma occurred at three postnatal weeks. The possible link of our data to the development of the Y visual processing stream and to the heterogeneity of the perigeniculate neuronal population is also discussed.

The Effects of Antipsychotics in Experimental Models of Krabbe Disease.

The role of altered myelin in the onset and development of schizophrenia and changes in myelin due to antipsychotics remains unclear. Antipsychotics are D2 receptor antagonists, yet D2 receptor agonists increase oligodendrocyte progenitor numbers and limit oligodendrocyte injury. Conflicting studies suggest these drugs promote the differentiation of neural progenitors to oligodendrocyte lineage, while others report antipsychotics inhibit the proliferation and differentiation of oligodendrocyte precursors. Here, we utilised in-vitro (human astrocytes), ex-vivo (organotypic slice cultures) and in-vivo (twitcher mouse model) experimental study designs of psychosine-induced demyelination, a toxin that accumulates in Krabbe disease (KD), to investigate direct effects of antipsychotics on glial cell dysfunction and demyelination. Typical and atypical antipsychotics, and selective D2 and 5HT2A receptor antagonists, attenuated psychosine-induced cell viability, toxicity, and morphological aberrations in human astrocyte cultures. Haloperidol and clozapine reduced psychosine-induced demyelination in mouse organotypic cerebellar slices. These drugs also attenuated the effects of psychosine on astrocytes and microglia and restored non-phosphorylated neurofilament levels, indicating neuroprotective effects. In the demyelinating twitcher mouse model of KD, haloperidol improved mobility and significantly increased the survival of these animals. Overall, this study suggests that antipsychotics directly regulate glial cell dysfunction and exert a protective effect on myelin loss. This work also points toward the potential use of these pharmacological agents in KD.

Cortical Layer Markers Expression and Increased Synaptic Density in Interstitial Neurons of the White Matter from Drug-Resistant Epilepsy Patients.

The interstitial neurons in the white matter of the human and non-human primate cortex share a similar developmental origin with subplate neurons and deep-layer cortical neurons. A subset of interstitial neurons expresses the molecular markers of subplate neurons, but whether interstitial neurons express cortical layer markers in the adult human brain remains unexplored. Here we report the expression of cortical layer markers in interstitial neurons in the white matter of the adult human brain, supporting the hypothesis that interstitial neurons could be derived from cortical progenitor cells. Furthermore, we found increased non-phosphorylated neurofilament protein (NPNFP) expression in interstitial neurons in the white matter of drug-resistant epilepsy patients. We also identified the expression of glutamatergic and g-aminobutyric acid (GABAergic) synaptic puncta that were distributed in the perikarya and dendrites of interstitial neurons. The density of glutamatergic and GABAergic synaptic puncta was increased in interstitial neurons in the white matter of drug-resistant epilepsy patients compared with control brain tissues with no history of epilepsy. Together, our results provide important insights of the molecular identity of interstitial neurons in the adult human white matter. Increased synaptic density of interstitial neurons could result in an imbalanced synaptic network in the white matter and participate as part of the epileptic network in drug-resistant epilepsy.

Insulin-like Growth Factor-1 Prevents Hypoxia/Reoxygenation-Induced White Matter Injury in Sickle Cell Mice

Occlusion of cerebral blood vessels causes acute cerebral hypoxia-an important trigger of ischemic white matter injury and stroke in sickle cell disease (SCD). While chronic hypoxia triggers compensatory neuroprotection via insulin-like growth factor-1 (IGF-1) and hypoxia inducible factor-1α (HIF-1α), severe bouts of acute hypoxia and subsequent restoration of blood flow (hypoxia/reoxygenation, H/R) overwhelm compensatory mechanisms and cause neuroaxonal damage-identified as white matter lesions-in the brain. The neuroprotective role of IGF-1 in the pathogenesis of white matter injury in SCD has not been investigated; however, it is known that systemic IGF-1 is reduced in individuals with SCD. We hypothesized that IGF-1 supplementation may prevent H/R-induced white matter injury in SCD. Transgenic sickle mice homozygous for human hemoglobin S and exposed to H/R developed white matter injury identified by elevated expression of non-phosphorylated neurofilament H (SMI32) with a concomitant decrease in myelin basic protein (MBP) resulting in an increased SMI32/MBP ratio. H/R-challenge also lowered plasma and brain IGF-1 expression. Human recombinant IGF-1 prophylaxis significantly induced HIF-1α and averted H/R-induced white matter injury in the sickle mice compared to vehicle-treated mice. The expression of the IGF-1 binding proteins IGFBP-1 and IGFBP-3 was elevated in the IGF-1-treated brain tissue indicating their potential role in mediating neuroprotective HIF-1α signaling. This study provides proof-of-concept for IGF-1-mediated neuroprotection in SCD.

Neuregulin-1/PI3K signaling effects on oligodendrocyte proliferation, remyelination and behaviors deficit in a male mouse model of ischemic stroke

In this study, we investigated the effect of neuregulin-1 (NRG1) on demyelination and neurological function in an ischemic stroke model, and further explored its neuroprotective mechanisms. Adult male ICR mice underwent photothrombotic ischemia surgery and were injected with NRG1 beginning 30 min after ischemia. Cylinder and grid walking tests were performed to evaluate the forepaw function. In addition, the effect of NRG1 on neuronal damage/death (Cresyl violet, CV), neuronal nuclei (NeuN), nestin, doublecortin (DCX), myelin basic protein (MBP), non-phosphorylated neurofilaments (SMI-32), adenomatous polyposis coli (APC), erythroblastic leukemia viral oncogene homolog (ErbB) 2, 4 and serine-threonine protein kinase (Akt) in cortex were evaluated using immunohistochemistry, immunofluorescence and western blot. The cylinder and grid walking tests exposed that treatment of NRG1 observably regained the forepaw function. NRG1 treatment reduced cerebral infarction, restored forepaw function, promoted proliferation and differentiation of neuron and increased oligodendrogliogenesis. The neuroprotective effect of NRG1 is involved in its activation of PI3K/Akt signaling pathway via ErbB2, as shown by the suppression of the effect of NRG1 by the PI3K inhibitor LY294002. Our results demonstrate that NRG1 is effective in ameliorating the both acute phase neuroprotection and long-term neurological functions via resumption of neuronal proliferation and differentiation and oligodendrogliogenesis in a male mouse model of ischemic stroke.

Neurochemical atlas of the cat spinal cord.

The spinal cord is a complex heterogeneous structure, which provides multiple vital functions. The precise surgical access to the spinal regions of interest requires precise schemes for the spinal cord structure and the spatial relation between the spinal cord and the vertebrae. One way to obtain such information is a combined anatomical and morphological spinal cord atlas. One of the widely used models for the investigation of spinal cord functions is a cat. We create a single cell-resolution spinal cord atlas of the cat using a variety of neurochemical markers [antibodies to NeuN, choline acetyltransferase, calbindin 28 kDa, calretinin, parvalbumin, and non-phosphorylated heavy-chain neurofilaments (SMI-32 antibody)] allowing to visualize several spinal neuronal populations. In parallel, we present a map of the spatial relation between the spinal cord and the vertebrae for the entire length of the spinal cord.

Purkinje cell vulnerability induced by diffuse traumatic brain injury is linked to disruption of long-range neuronal circuits

Cerebellar dysfunction is commonly observed following traumatic brain injury (TBI). While direct impact to the cerebellum by TBI is rare, cerebellar pathology may be caused by indirect injury via cortico-cerebellar pathways. To address the hypothesis that degeneration of Purkinje cells (PCs), which constitute the sole output from the cerebellum, is linked to long-range axonal injury and demyelination, we used the central fluid percussion injury (cFPI) model of widespread traumatic axonal injury in mice. Compared to controls, TBI resulted in early PC loss accompanied by alterations in the size of pinceau synapses and levels of non-phosphorylated neurofilament in PCs. A combination of vDISCO tissue clearing technique and immunohistochemistry for vesicular glutamate transporter type 2 show that diffuse TBI decreased mossy and climbing fiber synapses on PCs. At 2 days post-injury, numerous axonal varicosities were found in the cerebellum supported by fractional anisotropy measurements using 9.4 T MRI. The disruption and demyelination of the cortico-cerebellar circuits was associated with poor performance of brain-injured mice in the beam-walk test. Despite a lack of direct input from the injury site to the cerebellum, these findings argue for novel long-range mechanisms causing Purkinje cell injury that likely contribute to cerebellar dysfunction after TBI.

Axon hyperexcitability in the contralateral projection following unilateral optic nerve crush in mice.

Optic neuropathies are characterized by degeneration of retinal ganglion cell axonal projections to the brain, including acute conditions like optic nerve trauma and progressive conditions such as glaucoma. Despite different aetiologies, retinal ganglion cell axon degeneration in traumatic optic neuropathy and glaucoma share common pathological signatures. We compared how early pathogenesis of optic nerve trauma and glaucoma influence axon function in the mouse optic projection. We assessed pathology by measuring anterograde axonal transport from retina to superior colliculus, current-evoked optic nerve compound action potential and retinal ganglion cell density 1 week following unilateral optic nerve crush or intraocular pressure elevation. Nerve crush reduced axon transport, compound axon potential and retinal ganglion cell density, which were unaffected by intraocular pressure elevation. Surprisingly, optic nerves contralateral to crush demonstrated 5-fold enhanced excitability in compound action potential compared with naïve nerves. Enhanced excitability in contralateral sham nerves is not due to increased accumulation of voltage-gated sodium channel 1.6, or ectopic voltage-gated sodium channel 1.2 expression within nodes of Ranvier. Our results indicate hyperexcitability is driven by intrinsic responses of αON-sustained retinal ganglion cells. We found αON-sustained retinal ganglion cells in contralateral, sham and eyes demonstrated increased responses to depolarizing currents compared with those from naïve eyes, while light-driven responses remained intact. Dendritic arbours of αON-sustained retinal ganglion cells of the sham eye were like naïve, but soma area and non-phosphorylated neurofilament H increased. Current- and light-evoked responses of sham αOFF-sustained retinal ganglion cells remained stable along with somato-dendritic morphologies. In retinas directly affected by crush, light responses of αON- and αOFF-sustained retinal ganglion cells diminished compared with naïve cells along with decreased dendritic field area or branch points. Like light responses, αOFF-sustained retinal ganglion cell current-evoked responses diminished, but surprisingly, αON-sustained retinal ganglion cell responses were similar to those from naïve retinas. Optic nerve crush reduced dendritic length and area in αON-sustained retinal ganglion cells in eyes ipsilateral to injury, while crush significantly reduced dendritic branching in αOFF-sustained retinal ganglion cells. Interestingly, 1 week of intraocular pressure elevation only affected αOFF-sustained retinal ganglion cell physiology, depolarizing resting membrane potential in cells of affected eyes and blunting current-evoked responses in cells of saline-injected eyes. Collectively, our results suggest that neither saline nor sham surgery provide a true control, chronic versus acute optic neuropathies differentially affect retinal ganglion cells composing the ON and OFF pathways, and acute stress can have near-term effects on the contralateral projection.

Neurogenesis and neuronal differentiation in the postnatal frontal cortex in Down syndrome.

Although Down syndrome (DS), the most common developmental genetic cause of intellectual disability, displays proliferation and migration deficits in the prenatal frontal cortex (FC), a knowledge gap exists on the effects of trisomy 21 upon postnatal cortical development. Here, we examined cortical neurogenesis and differentiation in the FC supragranular (SG, II/III) and infragranular (IG, V/VI) layers applying antibodies to doublecortin (DCX), non-phosphorylated heavy-molecular neurofilament protein (NHF, SMI-32), calbindin D-28K (Calb), calretinin (Calr), and parvalbumin (Parv), as well as β-amyloid (APP/Aβ and Aβ1–42) and phospho-tau (CP13 and PHF-1) in autopsy tissue from age-matched DS and neurotypical (NTD) subjects ranging from 28-weeks (wk)-gestation to 3 years of age. Thionin, which stains Nissl substance, revealed disorganized cortical cellular lamination including a delayed appearance of pyramidal cells until 44 wk of age in DS compared to 28 wk in NTD. SG and IG DCX-immunoreactive (-ir) cells were only visualized in the youngest cases until 83 wk in NTD and 57 wk DS. Strong SMI-32 immunoreactivity was observed in layers III and V pyramidal cells in the oldest NTD and DS cases with few appearing as early as 28 wk of age in layer V in NTD. Small Calb-ir interneurons were seen in younger NTD and DS cases compared to Calb-ir pyramidal cells in older subjects. Overall, a greater number of Calb-ir cells were detected in NTD, however, the number of Calr-ir cells were comparable between groups. Diffuse APP/Aβ immunoreactivity was found at all ages in both groups. Few young cases from both groups presented non-neuronal granular CP13 immunoreactivity in layer I. Stronger correlations between brain weight, age, thionin, DCX, and SMI-32 counts were found in NTD. These findings suggest that trisomy 21 affects postnatal FC lamination, neuronal migration/neurogenesis and differentiation of projection neurons and interneurons that likely contribute to cognitive impairment in DS.

Abstract WP260: The Response Of Annexin A1 In Astrocyte Activation And White Matter Injury- A New Target For Stroke In Sickle Cell Disease

Introduction: Ischemic stroke is common in sickle cell disease (SCD). White matter injuries, incorporated into silent cerebral infarcts, are major risk factors of stroke in SCD. The pathogenesis of cerebral infarction in SCD is not known. Induced thrombogenesis may cause impaired astrocyte that stimulates demyelination leading to white matter injury. Annexin A1 (Anxa1) can regulate astrocyte function by inhibiting intracellular phospholipase A2 (PLA2). We hypothesized that astrocyte activation during thrombogenic events promotes white matter injury in SCD. We tested astrocyte dysfunction and white matter injury in a clinically relevant mouse model of SCD. Methods: Cerebral Infarctions induced by de novo thrombogenesis were identified by TTC staining in transgenic sickle (SS) and control (AA) mice with human sickle and normal hemoglobin respectively. Cerebral microvessels and astrocytes were isolated from SS and AA mice. Elisa assays were used to measure thrombin, PLA2 and Anxa1. Immunofluorescence staining was performed to determine astrocyte activation and demyelination. Results: Thrombogenesis within cerebral microvessels was elevated by ~2-fold (n=5, p<0.05), and cerebral infarction became prevalent in SS mice compared to AA mice (n=7, p<0.001). The infarcted brain tissue from SS mice showed neuronal cell death, whereas the brain tissue from the AA mice appeared normal. The SS mice had increased glial fibrillary acid protein positive (GFAP + ) activated astrocytes with significant increase in PLA2 accumulation. Relative abundance of non-phosphorylated neurofilament H (SMI32) and myelin basic protein (MBP) (SMI32/MBP), indicating white matter injury, was substantially induced in the SS compared to the AA. Expression of Anxa1 reduced significantly in plasma as well as within astrocytes’ end feet attached to the isolated cerebral microvessels of the SS mice. Recombinant Anxa1 prophylaxis to SS mice reduced the GFAP + astrocytes, PLA2 accumulation, demyelination and infarct score (n=6, p<0.01) compared to SS mice treated with vehicle. Conclusion: White matter injury in SS mice is associated with astrocyte dysfunction and lack of Anxa1, which may be developed as potential therapeutic targets to prevent and/or treat stroke in SCD.

Specific neuronal subpopulations in the amygdala of macaque monkeys express high levels of nonphosphorylated neurofilaments

Pyramidal neurons in the neocortex that express nonphosphorylated neurofilaments (NPNFs) are especially vulnerable to degeneration in Alzheimer’s disease. Since the basolateral nuclear complex of the amygdala (BNC) and cortical nuclear complex of the amygdala (CNC) are cortex-like structures, containing both pyramidal (PNs) and nonpyramidal neurons (NPNs), it is of interest to determine which cell types in the primate BNC and CNC are NPNF+. We also studied NPNF expression in the non-cortex-like nuclei of the amygdala (central and medial nuclei). Digitized images of sections through fetal, newborn, infant, and adult macaque brains stained for NPNFs, obtained from the Macaque Brain Resource (MacBrainResource, MBR), were analyzed. The pattern of NPNF immunoreactivity (NPNF-ir) in the BNC, CNC, and medial nucleus was essentially identical in all four age groups, but there were some age-dependent differences in the central nucleus. All BNC and CNC nuclei contained a moderate density of NPNF+ NPNs. Both the somata and the entire dendritic arborizations of these NPNs were stained. PNs with robust NPNF-ir in their somata and proximal dendrites were only seen in the basal magnocellular nucleus, where it appeared that virtually every PN was NPNF+. This pattern of NPNF expression is distinct from that seen in the mammalian neocortex, where NPNF+ neurons are almost entirely PNs, but is very similar to that seen in a recent study of the rat BNC. These findings, in conjunction with the cortical data, suggest the possibility that NPNF+ neuronal subpopulations in the BNC and CNC might be especially vulnerable in Alzheimer’s disease.

SMI-32 labeling in Cajal-Retzius cells of feline primary visual cortex

Cajal-Retzius cells are one of the transient elements of the developing cerebral cortex. These cells express some characteristic molecules. One of them, heavy-chain neurofilaments, participating in the construction of the mature cerebral networks, are believed to be a specific feature of the human’s Cajal-Retzius cells. Using histochemical stain for SMI-32 antibody to the non-phosphorylated heavy-chain neurofilaments, large neurons having horizontally oriented soma and bipolar processes were labeled in the molecular layer of the primary visual cortex of cats aged 0–2 postnatal days. Using DiI technique, similar neurons having a well-developed system of parallel vertical branches coming from the two horizontal processes were visualized in these areas. The location and general morphology of these neurons were similar to the Cajal-Retzius cells allowing to suppose for the carnivores to share similar with primates developmental mechanisms of the corticogenesis.

P 25. Investigating cytoskeletal integrity in the sensory nerve fibers in healthy individuals

Introduction. Neurons with long neurites are physiologically remarkable, as they need to transport newly synthesized proteins and organelles (e.g. mitochondria) from the soma over long distances to the synapses, which is essential for neuronal homeostasis. Via adaptor proteins, which walk on microtubules, organelles find their target place. Next to microtubules, the neuronal cytoskeleton consists of neurofilaments, and actin: neurofilaments regulate axonal caliber, and actin is involved in the neuronal shape (Eira et al. 2016, Hoffman et al. 1984). A misbalance or dysfunction of cytoskeletal components might contribute to neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS). Neurofilament light chain (NfL) is an established biomarker for ALS (Bacioglu et al. 2016). Here, we study the human cytoskeletal components during aging . As a model we chose sensory neurons which possess long processes and axon endings can be accessed easily by skin biopsies. In this study we focus on the expression levels of microtubules, neurofilaments, and actin in skin biopsies of healthy individuals. Furthermore, we analyze the axonal diameter. Methods. Skin biopsies (thigh and ankle) of 27 men (mean age 54.2 ± 18.9 a, from 24 a to 79 a) were PFA-fixed and cut. Fluorescent immunostaining for non-phosphorylated neurofilament heavy chain (npNfH), betaIII-tubulin, and actin was performed. Images of the dermis were acquired by confocal microscopy. At least 20 axons per healthy individual were analyzed for the mean gray values and diameter. Statistics were calculated using GraphPad Prism (V9). This study was approved by the local ethics committee. Participants gave informed written consent. Results. Cytoskeletal components (npNfH, betaIII-tubulin, actin) drastically increased with age. NpNfH increased 2.7-fold from 24- to 75-year-old individuals. Actin showed a change of 3.8-fold from the youngest to the 62-year-old controls (peak, maximum values), whereas betaIII-tubulin levels increased 3.2-fold from the 24- to the 75-year-old healthy individuals. Sensory axon caliber increased 2.2-fold from the 29- (1.35 µm) to the 78/79-year old (2.97 µm) controls which results in a 4.9-fold increase of the cross-sectional area of the axons. Statistical analysis (Mann-Whitney U test) showed highly significant results between each staining/caliber and age ( p < 0.001). Discussion. Our results suggest an age-driven “ossification” of the axonal cytoskeleton in peripheral sensory axons, which might influence cytoskeletal functionality and axonal caliber. The age-dependent increase in caliber can be explained by the higher expression of neurofilaments, which mainly regulate the diameter of axons (Costa et al. 2018). These findings are in accordance with the increased susceptibility of aged individuals to neurodegenerative diseases. The results reported here will be validated in a second independent cohort (27 men, age 26 to 74). We further plan the analysis of length dependency as biopsies were obtained from thigh and ankle.

Specific neuronal subpopulations in the rat basolateral amygdala express high levels of nonphosphorylated neurofilaments.

Cortical pyramidal neurons (PNs) containing nonphosphorylated neurofilaments (NNFs) localized with the SMI-32 monoclonal antibody have been shown to be especially vulnerable to degeneration in Alzheimer's disease (AD). The present investigation is the first to study the expression of SMI-32+ NNFs in neurons of the basolateral nuclear complex of the amygdala (BNC), which contains cortex-like PNs and nonpyramidal neurons (NPNs). We observed that PNs in the rat basolateral nucleus (BL), but not in the lateral (LAT) or basomedial (BM) nuclei, have significant levels of SMI-32-ir in their somata with antibody diluents that did not contain Triton X-100, but staining in these cells was greatly attenuated when the antibody diluent contained 0.3% Triton. Using Triton-containing diluents, we found that all SMI-32+ neurons in all three of the BNC nuclei were NPNs. Using a dual-labeling immunoperoxidase technique, we demonstrated that most of these SMI-32+ NPNs were parvalbumin-positive (PV+) or somatostatin-positive NPNs but not vasoactive intestinal peptide-positive or neuropeptide Y-positive NPNs. Using a technique that combines retrograde tracing with SMI-32 immunohistochemistry using intermediate levels of Triton in the diluent, we found that all BNC neurons projecting to the mediodorsal thalamic nucleus (MD) were large NPNs, and most were SMI-32+. In contrast, BNC neurons projecting to the ventral striatum or cerebral cortex were PNs that expressed low levels of SMI-32 immunoreactivity (SMI-32-ir) in the BL, and no SMI-32-ir in the LAT or BM. These data suggest that the main neuronal subpopulations in the BNC that degenerate in AD may be PV+ and MD-projecting NPNs.

SMI-32 — a novel axonal injury marker for investigation of ischemic brain pathology

The relevance of this work is determined by the high prevalence and social significance of cerebrovascular diseases and the need to develop effective methods for verifying neuronal damage due to cerebral ischemia in experimental models.&#x0D; The aim of this study was to assess the possibility of immunohistochemical revealing of neurofilaments to detect axonal injury in cerebral ischemia models.&#x0D; Materials and methods. A model of transient focal cerebral ischemia by the left middle cerebral artery occlusion was reproduced in male Wistar, SHR and WKY rats. Axonal injury was assessed by immunohistochemical reactions for neurofilament proteins using SMI-32 and 2F11 antibodies.&#x0D; Results. In cerebral ischemia, damage to nerve fibers occurs, manifested by thickening of axons, their varicose expansion and segmental accumulation of neurofilament proteins. These changes are more noticeable with an immunohistochemical reaction to the SMI-32 marker of neurofilament heavy chain.&#x0D; Conclusions. The use of antibodies to the non-phosphorylated neurofilament heavy chain makes it easy to identify degenerating nerve fibers and can be recommended as an alternative method for detecting axonal injury.

Cerebellar Calcium-Binding Protein and Neurotrophin Receptor Defects in Down Syndrome and Alzheimer's Disease.

Cerebellar hypoplasia is a major characteristic of the Down syndrome (DS) brain. However, the consequences of trisomy upon cerebellar Purkinje cells (PC) and interneurons in DS are unclear. The present study performed a quantitative and qualitative analysis of cerebellar neurons immunostained with antibodies against calbindin D-28k (Calb), parvalbumin (Parv), and calretinin (Calr), phosphorylated and non-phosphorylated intermediate neurofilaments (SMI-34 and SMI-32), and high (TrkA) and low (p75NTR) affinity nerve growth factor (NGF) receptors as well as tau and amyloid in DS (n = 12), Alzheimer's disease (AD) (n = 10), and healthy non-dementia control (HC) (n = 8) cases. Our findings revealed higher Aβ42 plaque load in DS compared to AD and HC but no differences in APP/Aβ plaque load between HC, AD, and DS. The cerebellar cortex neither displayed Aβ40 containing plaques nor pathologic phosphorylated tau in any of the cases examined. The number and optical density (OD) measurements of Calb immunoreactive (-ir) PC soma and dendrites were similar between groups, while the number of PCs positive for Parv and SMI-32 were significantly reduced in AD and DS compared to HC. By contrast, the number of SMI-34-ir PC dystrophic axonal swellings, termed torpedoes, was significantly greater in AD compared to DS. No differences in SMI-32- and Parv-ir PC OD measurements were observed between groups. Conversely, total number of Parv- (stellate/basket) and Calr (Lugaro, brush, and Golgi)-positive interneurons were significantly reduced in DS compared to AD and HC. A strong negative correlation was found between counts for Parv-ir interneurons, Calr-ir Golgi and brush cells, and Aβ42 plaque load. Number of TrkA and p75NTR positive PCs were reduced in AD compared to HC. These findings suggest that disturbances in calcium binding proteins play a critical role in cerebellar neuronal dysfunction in adults with DS.