Biology subjects

Richardson, J. R.

Publications and source records attributed to Richardson, J. R..

8 recordsLinked to original sources

bioRxiv2025

A 3D Human Neuron-on-Chip Platform to Monitor Neuronal Injury Responses

Traumatic brain injury (TBI) is a major cause of neurological dysfunction and long-term neurodegeneration, yet the intrinsic neuronal contributions to TBI pathophysiology remain incompletely defined. Here, we present a novel Neuron-on-Chip microfluidic platform that can be used to mechanically injure mature human prefrontal cortex neurons (hPFCs) embedded in three-dimensional (3D) hydrogels, enabling the study of injury responses in pure neuronal cultures. We assessed real-time calcium dynamics across 13 metrics of single-cell and network activity, revealing a biphasic injury response: an early phase (0.5-24 hr) characterized by excitotoxicity, hyper-synchronized bursting, and network collapse; and a late phase (8 d) marked by sustained depolarization and structural remodeling. Secretome profiling uncovered progressive elevations in extracellular pT181 and total Tau from days 1 to 5 post-injury. Cytokine analyses identified early (24 hr) elevations in IP-10, IL-10, IFN2, and NCAM, and late increases (8 d) in CXCL9 and MPO, linking neuronal activity changes to stage-specific inflammatory signaling. Immunocytochemistry and immunoblotting confirmed temporally ordered upregulation of calpain-1 and active caspase-3 (days 1-3), phosphorylated Tau (AT8+, days 5-8), and neurofibrillary tangle-like Tau aggregates (NFT+, day 8). These findings establish our platform as a scalable microphysiological model for probing the dynamic cellular and molecular sequelae of neuronal response to injury, offering insights into neurodegeneration and opportunities for therapeutic discovery.

neuroscience↗

bioRxiv2025

Effects of Pesticide Exposure on Neuroinflammation and Microglial Gene Expression: Relevance to Mechanisms of Alzheimer's Disease Risk

BackgroundAlzheimers disease (AD) is characterized by the presence of amyloid-{beta} plaques, neurofibrillary tangles, and neuroinflammation. Previously, we reported serum levels of dichlorodiphenyldichloroethylene (DDE), the primary metabolite of the pesticide dichlorodiphenyltrichloroethane (DDT), were significantly higher in AD patients compared to age-matched controls and that DDT exposure worsened AD pathology in animal models. ObjectiveHere, we investigated the effect of DDT on neuroinflammation in primary mouse microglia (PMG) and C57BL/6J mice. MethodsEffects of DDT on inflammation and disease-associated microglia were determined in primary mouse microglia and C57BL/6J mice. ResultsPMG exposed to DDT (0.5-5.0 {micro}M) elicited a [~]2-3-fold increase in Il-1b mRNA levels, with similar concentration-dependent upregulation in Il-6, Nos2, and Tnfa. These effects were blocked by the sodium channel antagonist tetrodotoxin, demonstrating the role of DDT-microglial sodium channel interactions in mediating this response. Additionally, NOS2 protein levels increased by [~]1.5-2-fold, while TNFa was elevated by 2-4-fold. C57BL/6J male and female mice exposed to DDT (30 mg/kg) demonstrated significantly increased mRNA levels of Nos2, Il-1b, and Il-6 in the frontal cortex (1.5-2.3-fold), and Nos2, Il-1b, and Tnfa (1.5-1.8-fold) in the hippocampus. Furthermore, microglial homeostatic genes, Cx3cr1, P2ry12, and Tmem119, were downregulated, while stage 1 disease-associated microglia genes were upregulated both in vitro and in vivo. Notably, Apoe and Trem2 were only upregulated in the frontal cortex and hippocampus of females. ConclusionThese data indicate that DDT increases neuroinflammation, which may result from direct actions of DDT on microglia, providing a novel pathway by which DDT may contribute to AD risk.

neuroscience↗

bioRxiv2024

Physiological JNK3 Concentrations Are Higher in Motor-related and Disease-implicated Brain Regions of C57BL6/J Mice

The c-Jun N-terminal kinase 3 (JNK3) is a stress-responsive protein kinase primarily expressed in the central nervous system (CNS). JNK3 exhibits nuanced neurological activities, such as roles in behavior, circadian rhythms, and neurotransmission, but JNK3 is also implicated in cell death and neurodegeneration. Despite the critical role of JNK3 in neurophysiology and pathology, its localization in the brain is not fully understood due to a paucity of tools to distinguish JNK3 from other isoforms. While previous functional and histological studies suggest locales for JNK3 in the CNS, a comprehensive and higher resolution of JNK3 distribution and abundance remained elusive. Here, we sought to define the anatomical and cellular distribution of JNK3 in adult mouse brains. Data reveal the highest levels of JNK3 and pJNK3 were found in the cortex and the hippocampus. JNK3 possessed neuron-type selectivity as JNK3 was present in GABAergic, cholinergic, and dopaminergic neurons, but was not detectable in VGLUT-1-positive glutamatergic neurons and astrocytes in vivo. Intriguingly, higher JNK3 signals were found in motor neurons and relevant nuclei in the cortex, basal ganglia, brainstem, and spinal cord. While JNK3 was primarily observed in the cytosol of neurons in the cortex and the hippocampus, JNK3 appeared commonly within the nucleus in the brainstem. These distinctions suggest the potential for significant differences between JNK3 actions in distinct brain regions and cell types. Our results provide a significant improvement over previous reports of JNK3 spatial organization in the adult CNS and support continued investigation of JNK3s role in neurophysiology and pathophysiology.

neuroscience↗

bioRxiv2023

Moderate intensity aerobic exercise in 6-OHDA-lesioned rats alleviates established motor deficits and reduces neurofilament light and glial fibrillary acidic protein serum levels without increased striatal dopamine or tyrosine hydroxylase protein

BackgroundAlleviation of motor impairment by aerobic exercise (AE) in Parkinsons disease (PD) points to a CNS response that could be targeted by therapeutic approaches, but recovery of striatal dopamine (DA) or tyrosine hydroxylase (TH) has been inconsistent in rodent studies. ObjectiveTo increase translation of AE, 3 components were implemented into AE design to determine if recovery of established motor impairment, concomitant with >80% striatal DA and TH loss, was possible. We also evaluated if serum levels of neurofilament light (NfL) and glial fibrillary acidic protein (GFAP), blood-based biomarkers of disease severity in human PD, were affected. MethodsWe used a 6-OHDA hemiparkinson rat model featuring progressive nigrostriatal neuron loss over 28 days, with impaired forelimb use 7 days post-lesion, and hypokinesia onset 21 days post-lesion. After establishing forelimb use deficits, moderate intensity AE began 1-3 days later, 3x per week, for 40 min/session. Motor assessments were conducted weekly for 3 wks, followed by determination of striatal DA, TH protein and mRNA, and NfL and GFAP serum levels. ResultsSeven days after 6-OHDA lesion, recovery of depolarization-stimulated extracellular DA and DA tissue content was <10%, representing severity of DA loss in human PD, concomitant with 50% reduction in forelimb use. Despite severe DA loss, recovery of forelimb use deficits and alleviation of hypokinesia progression began after 2 weeks of AE and was maintained. Increased NfLand GFAP levels from lesion were reduced by AE. Despite these AE-driven changes, striatal DA tissue and TH protein levels were unaffected. ConclusionsThis proof-of-concept study shows AE, using exercise parameters within the capabilities most PD patients, promotes recovery of established motor deficits in a rodent PD model, concomitant with reduced levels of blood-based biomarkers associated with PD severity, without commensurate increase in striatal DA or TH protein.

neuroscience↗

bioRxiv2023

A partial Drp1 knockout improves autophagy flux independent of mitochondrial function

Dynamin-related protein 1 (Drp1) is typically known for its role in mitochondrial fission. A partial inhibition of this protein has been reported to be protective in experimental models of neurodegenerative diseases. The protective mechanism has been attributed primarily to improved mitochondrial function. Herein, we provide evidence showing that a partial Drp1-knockout improves autophagy flux independent of mitochondria. First, we characterized in cell and animal models that at low non-toxic concentrations, manganese (Mn), which causes parkinsonian-like symptoms in humans, impaired autophagy flux but not mitochondrial function and morphology. Furthermore, nigral dopaminergic neurons were more sensitive than their neighbouring GABAergic counterparts. Second, in cells with a partial Drp1-knockdown and Drp1+/- mice, autophagy impairment induced by Mn was significantly attenuated. This study demonstrates that autophagy is a more vulnerable target than mitochondria to Mn toxicity. Furthermore, improving autophagy flux is a separate mechanism conferred by Drp1 inhibition independent of mitochondrial fission.

cell biology↗

bioRxiv2023

Differential expression of RET and GDNF family receptor, GFR-α1, between striatum and substantia nigra following nigrostriatal lesion: a case for diminished GDNF-signaling

Although glial cell line-derived neurotrophic factor (GDNF) showed efficacy in preclinical and early clinical studies to alleviate parkinsonian signs in Parkinsons disease (PD), later trials did not meet primary endpoints, giving pause to consider further investigation. While GDNF dose and delivery methods may have contributed to diminished efficacy, one crucial aspect of these clinical studies is that GDNF treatment across all studies began [~]8 years after PD diagnosis; a time point representing several years after near 100% depletion of nigrostriatal dopamine markers in striatum and at least 50% in substantia nigra (SN), and is later than the timing of GDNF treatment in preclinical studies. With nigrostriatal terminal loss exceeding 70% at PD diagnosis, we utilized hemi-parkinsonian rats to determine if expression of GDNF family receptor, GFR-1, and receptor tyrosine kinase, RET, differed between striatum and SN at 1 and 4 weeks following a 6-hydroxydopamine (6-OHDA) lesion. Whereas GDNF expression changed minimally, GFR-1 expression decreased progressively in striatum and in tyrosine hydroxylase positive (TH+) cells in SN, correlating with reduced TH cell number. However, in nigral astrocytes, GFR-1 expression increased. RET expression decreased maximally in striatum by 1 week, whereas in the SN, a transient bilateral increase occurred that returned to control levels by 4 weeks. Expression of brain-derived neurotrophic factor (BDNF) or its receptor, TrkB, were unchanged throughout lesion progression. Together, these results reveal that differential GFR-1 and RET expression between the striatum and SN, and cell-specific differences in GFR-1 expression in SN, occur during nigrostriatal neuron loss. Targeting loss of GDNF receptors appears critical to enhance GDNF therapeutic efficacy against nigrostriatal neuron loss. Significance StatementAlthough preclinical evidence supports that GDNF provides neuroprotection and improves locomotor function in preclinical studies, clinical data supporting its efficacy to alleviate motor impairment in Parkinsons disease patients remains uncertain. Using the established 6-OHDA hemi-parkinsonian rat model, we determined whether expression of its cognate receptors, GFR-1 and RET, were differentially affected between striatum and substantia nigra in a timeline study. In striatum, there was early and significant loss of RET, but a gradual, progressive loss of GFR-1. In contrast, RET transiently increased in lesioned substantia nigra, but GFR-1 progressively decreased only in nigrostriatal neurons and correlated with TH cell loss. Our results indicate that direct availability of GFR-1 may be a critical element that determines GDNF efficacy following striatal delivery. HighlightsGDNF expression was minimally affected by nigrostriatal lesion GDNF family receptor, GFR-1, progressively decreased in striatum and in TH neurons in SN. GFR-1 expression decreased along with TH neurons as lesion progressed GFR-1 increased bilaterally in GFAP+ cells suggesting an inherent response to offset TH neuron loss RET expression was severely reduced in striatum, whereas it increased in SN early after lesion induction

neuroscience↗

bioRxiv2022

Nigral-specific increase in ser31 tyrosine hydroxylase phosphorylation offsets dopamine loss and forestalls hypokinesia onset during progressive nigrostriatal neuron loss

Mechanisms that augment dopamine (DA) signaling to compensate for tyrosine hydroxylase (TH) loss and delay motor impairment in Parkinsons disease remain unidentified. The rat nigrostriatal pathway was unilaterally-lesioned by 6-OHDA to determine whether differences in DA content, TH protein, TH phosphorylation, or D1 receptor expression in striatum or substantia nigra (SN) aligned with onset of hypokinesia at two time points. At 7 days, DA and TH loss in striatum exceeded 95%, whereas DA was unaffected in SN, despite [~]60% TH loss. At 28 days, hypokinesia was established. At both time points, ser31 TH phosphorylation increased only in SN, corresponding to less DA versus TH loss. ser40 TH phosphorylation was unaffected in striatum or SN. By day 28, D1 receptor expression increased only in lesioned SN. These results indicate that increased ser31 TH phosphorylation and D1 receptor in the SN, not striatum, augment DA signaling against TH loss to mitigate hypokinesia. Highlights-Despite >90% TH and DA loss in striatum, open-field locomotor activity did not decrease -Early after lesion, DA and TH loss in striatum exceeded 90%. In contrast, DA loss did not occur despite 60% TH loss in substantia nigra (SN). -TH loss was progressive in the SN, with loss also spreading contralateral to lesion. -Loss of TH protein in SN preceded cell loss ipsilateral and contralateral to lesion, indicating first stages of nigrostriatal neuron loss begin with loss of TH protein loss. -TH phosphorylation at ser31 in SN was associated with less, if any, DA loss compared to TH protein loss. -TH phosphorylation at ser40 did not change in either region and at any time during TH loss, suggesting no contribution to differences in DA loss against TH loss. -Expression of the D1 receptor increased 2.5-fold in the SN late, but not early, after lesion, suggesting a post-synaptic receptor response to offset DA loss in SN. -No increases in TH phosphorylation or D1 receptor expression in striatum at any time after lesion induction, indicating that compensatory mechanisms occur only in substantia nigra, but not in striatum, to delay onset of hypokinesia.

neuroscience↗

bioRxiv2018

Knockdown of butyrylcholinesterase but not inhibition by chlorpyrifos alters early differentiation mechanisms in human neural stem cells

Butyrylcholinesterase (BChE) is the evolutionary counterpart to acetylcholinesterase (AChE). Both are expressed early in nervous system development prior to cholinergic synapse formation. The organophosphate pesticide chlorpyrifos (CPF) primarily exerts toxicity through inhibition of AChE, which results in excess cholinergic stimulation at the synapse. We hypothesized that inhibition of AChE and BChE by CPF may impair early neurogenesis in neural stem cells (NSCs). To model neurodevelopment in vitro, we used human NSCs derived from induced pluripotent stem cells (iPSCs) with a focus on initial differentiation mechanisms. Over six days of NSC differentiation, BChE activity and mRNA expression significantly increased, while AChE activity and expression remained unchanged. CPF treatment (10 M) caused 82% and 92% inhibition of AChE and BChE, respectively. CPF exposure had no effect on cell viability or the expression of differentiation markers HES5, DCX or MAP2. However, shRNA-knockdown of BChE expression resulted in decreased or delayed expression of transcription factors HES5 and HES3. BChE may have a role in the differentiation of NSCs independent of, or in addition to, its enzymatic activity.

pharmacology and toxicology↗