Alexander M. Herman

Inflammation in the early stages of neurodegenerative pathology

Inflammation is secondary to protein accumulation in neurodegenerative diseases, including Alzheimers, Parkinsons and Amyotrophic Lateral Sclerosis. Emerging evidence indicate sustained inflammatory responses, involving microglia and astrocytes in animal models of neurodegeneration. It is unknown whether inflammation is beneficial or detrimental to disease progression and how inflammatory responses are induced within the CNS. Persistence of an inflammatory stimulus or failure to resolve sustained inflammation can result in pathology, thus, mechanisms that counteract inflammation are indispensable. Here we review studies on inflammation mediated by innate and adaptive immunity in the early stages of neurodegeneration and highlight important areas for future investigation.

Parkin mediates beclin-dependent autophagic clearance of defective mitochondria and ubiquitinated Aβ in AD models

Intraneuronal amyloid-β (Aβ) may contribute to extracellular plaque deposition, the characteristic pathology of Alzheimers disease (AD). The E3-ubiquitin ligase parkin ubiquitinates intracellular proteins and induces mitophagy. We previously demonstrated that parkin reduces Aβ levels in lentiviral models of intracellular Aβ. Here we used a triple transgenic AD (3xTg-AD) mouse, which over-expresses APP(Swe), Tau(P301L) and harbor the PS1(M146V) knock-in mutation and found that lentiviral parkin ubiquitinated intracellular Aβ in vivo, stimulated beclin-dependent molecular cascade of autophagy and facilitated clearance of vesicles containing debris and defective mitochondria. Parkin expression decreased intracellular Aβ levels and extracellular plaque deposition. Parkin expression also attenuated caspase activity, prevented mitochondrial dysfunction and oxidative stress and restored neurotransmitter synthesis. Restoration of glutamate synthesis, which was independent of glial-neuronal recycling, depended on mitochondrial activity and led to an increase in γ-amino butyric acid levels. These data indicate that parkin may be used as an alternative strategy to reduce Aβ levels and enhance autophagic clearance of Aβ-induced defects in AD. Parkin-mediated clearance of ubiquitinated Aβ may act in parallel with autophagy to clear molecular debris and defective mitochondria and restore neurotransmitter balance.

Cell type-specific and time-dependent light exposure contribute to silencing in neurons expressing Channelrhodopsin-2

Channelrhodopsin-2 (ChR2) has quickly gained popularity as a powerful tool for eliciting genetically targeted neuronal activation. However, little has been reported on the response kinetics of optogenetic stimulation across different neuronal subtypes. With excess stimulation, neurons can be driven into depolarization block, a state where they cease to fire action potentials. Herein, we demonstrate that light-induced depolarization block in neurons expressing ChR2 poses experimental challenges for stable activation of specific cell types and may confound interpretation of experiments when ‘activated’ neurons are in fact being functionally silenced. We show both ex vivo and in vivo that certain neuronal subtypes targeted for ChR2 expression become increasingly susceptible to depolarization block as the duration of light pulses are increased. We find that interneuron populations have a greater susceptibility to this effect than principal excitatory neurons, which are more resistant to light-induced depolarization block. Our results highlight the need to empirically determine the photo-response properties of targeted neurons when using ChR2, particularly in studies designed to elicit complex circuit responses in vivo where neuronal activity will not be recorded simultaneous to light stimulation. DOI: http://dx.doi.org/10.7554/eLife.01481.001

β-amyloid triggers ALS-associated TDP-43 pathology in AD models.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease associated with loss of motor neurons in the brain and spinal cord. ALS is occasionally diagnosed with frontotemporal lobar dementia with ubiquitin-positive inclusions (FTLD-U). Alzheimers disease (AD) is the most common type of age-associated dementia. Abnormal levels of aggregated Tar-DNA binding protein-43 (TDP-43) are detected in the majority of patients with ALS, FTLD and AD. We observed a significant increase (200%) in the levels of TDP-43 in cortical autopsies of late stage AD patients. Lentiviral expression of Aβ(1-42) in the rat motor cortex led to an increase in TDP-43 pathology, including up-regulation of the mature ~44kDa protein, identical to the pathological changes seen in AD. Furthermore, expression of Aβ(1-42) was associated with TDP-43 phosphorylation and accumulation in the cytosol. Clearance of Aβ with parkin prevented TDP-43 pathology. TDP-43 modifications were also observed in 3xTransgenic AD (3xTg-AD) compared to wild type mice, but these changes were attenuated in parkin-injected hippocampi, even in the presence of Tau pathology, suggesting that TDP-43 pathology is triggered by Aβ, independent of Tau. Increased levels of casein kinase (CK1 and CK2), which are associated with TDP-43 phosphorylation, were also observed in Aβ(1-42) expressing brains. These data indicate an overlap in TDP-43 pathology between AD and ALS-FTLD and suggest that Aβ triggers modifications of TDP-43.

Wild type and P301L mutant Tau promote neuro-inflammation and α-Synuclein accumulation in lentiviral gene delivery models.

Neurodegeneration involves multiple pathogenic proteins, including Tau, Aβ, TDP-43 and α-Synuclein, but there is little information how these pathogenic proteins interact. We cloned human wild type 4 repeat Tau (Tau(wt)) and mutant Tau(P301L) into a lentivirus and performed stereotaxic injection into the rat motor cortex to examine Tau modification, neuro-inflammation and changes of other proteins associated with neurodegeneration. Tau(P301L) was associated with more phosphorylation of Tau, including Thr 181 and Ser 262 residues and resulted in more aggregation. Both forms of Tau expression increased glycogen synthase kinase-3 (GSK-3) activity, polo-like kinase-2 (PLK2) levels and decreased protein phosphatase activity, but had no effects on casein kinase-1 (CK1). No changes were observed in glial fibrillary acidic protein (GFAP) staining with either Tau(wt) or Tau(P301L), but both caused microglial changes and higher interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels. Tau(wt) and Tau(P301L) increased the levels of endogenous α-Synuclein, but not β-amyloid precursor protein (βAPP) or Tar-DNA binding protein (TDP-43). The levels of phosphorylated Ser-129 α-Synuclein (p-Ser129) were also increased with Tau(wt) and Tau(P301L) expressing animals. These data suggest that Tau(wt) and Tau(P301L) alter kinase activities, but they differentially induce inflammation, Tau modification and α-Synuclein phosphorylation. This change of α-Synuclein in Tau gene transfer models suggests that Tau pathology may lead to α-Synuclein modification in neurodegenerative diseases.

A cholinergic basal forebrain feeding circuit modulates appetite suppression

Atypical food intake is a primary cause of obesity and other eating and metabolic disorders. Insight into the neural control of feeding has previously focused mainly on signalling mechanisms associated with the hypothalamus, the major centre in the brain that regulates body weight homeostasis. However, roles of non-canonical central nervous system signalling mechanisms in regulating feeding behaviour have been largely uncharacterized. Acetylcholine has long been proposed to influence feeding owing in part to the functional similarity between acetylcholine and nicotine, a known appetite suppressant. Nicotine is an exogenous agonist for acetylcholine receptors, suggesting that endogenous cholinergic signalling may play a part in normal physiological regulation of feeding. However, it remains unclear how cholinergic neurons in the brain regulate food intake. Here we report that cholinergic neurons of the mouse basal forebrain potently influence food intake and body weight. Impairment of cholinergic signalling increases food intake and results in severe obesity, whereas enhanced cholinergic signalling decreases food consumption. We found that cholinergic circuits modulate appetite suppression on downstream targets in the hypothalamus. Together our data reveal the cholinergic basal forebrain as a major modulatory centre underlying feeding behaviour.

Local corticotropin releasing hormone (CRH) signals to its receptor CRHR1 during postnatal development of the mouse olfactory bulb

Neuropeptides play important physiological functions during distinct behaviors such as arousal, learning, memory, and reproduction. However, the role of local, extrahypothalamic neuropeptide signaling in shaping synapse formation and neuronal plasticity in the brain is not well understood. Here, we characterize the spatiotemporal expression profile of the neuropeptide corticotropin-releasing hormone (CRH) and its receptor CRHR1 in the mouse OB throughout development. We found that CRH-expressing interneurons are present in the external plexiform layer, that its cognate receptor is expressed by granule cells, and show that both CRH and CRHR1 expression enriches in the postnatal period when olfaction becomes important towards olfactory-related behaviors. Further, we provide electrophysiological evidence that CRHR1-expressing granule cells functionally respond to CRH ligand, and that the physiological circuitry of CRHR1 knockout mice is abnormal, leading to impaired olfactory behaviors. Together, these data suggest a physiologically relevant role for local CRH signaling towards shaping the neuronal circuitry within the mouse OB.

Wild type TDP-43 induces neuro-inflammation and alters APP metabolism in lentiviral gene transfer models.

The transactivation DNA-binding protein (TDP-43) pathology is associated with fronto-temporal lobar dementia (FTLD) with ubiquitinated inclusions and some cases of Alzheimers disease (AD). Proteolytic fragments of β-amyloid precursor protein (βAPP) are detected in AD as well as the cerebrospinal fluid (CSF) from FTLD and Amyotrophic Lateral Sclerosis (ALS) patients, suggesting alteration in APP processing. Because of the overlap in TDP-43 pathology between FTLD and AD, we sought to determine whether there is a relationship between TDP-43 and APP metabolism. We generated gene transfer models using lentiviral delivery of human TDP-43 and Aβ(1-42) into the rat primary motor cortex and examined their role 2 weeks post-injection. Expression of TDP-43 and/or Aβ(1-42) increase pro-inflammatory markers, including Interleukin (IL)-6, tumor necrosis factor (TNF-α), glial neurofibrillary proteins (GFAP) and ionized calcium binding adaptor molecule 1 (IBA-1). Lentiviral Aβ(1-42) up-regulates endogenous TDP-43 and promotes its phosphorylation, aggregation and cleavage into 35 kDa fragments. Inversely, lentiviral TDP-43 expression increases the levels and activity of β-secretase (BACE), accelerating production of APP C-terminal fragments (C99) and Aβ(1-40). Here we show that TDP-43 up-regulates APP metabolism and suggest a mechanistic link between TDP-43 and BACE.

Local CRH signaling promotes synaptogenesis and circuit integration of adult-born neurons

Neural activity either enhances or impairs de novo synaptogenesis and circuit integration of neurons, but how this activity is mechanistically relayed in the adult brain is largely unknown. Neuropeptide-expressing interneurons are widespread throughout the brain and are key candidates for conveying neural activity downstream via neuromodulatory pathways that are distinct from classical neurotransmission. With the goal of identifying signaling mechanisms that underlie neuronal circuit integration in the adult brain, we have virally traced local corticotropin-releasing hormone (CRH)-expressing inhibitory interneurons with extensive presynaptic inputs onto new neurons that are continuously integrated into the adult rodent olfactory bulb. Local CRH signaling onto adult-born neurons promotes and/or stabilizes chemical synapses in the olfactory bulb, revealing a neuromodulatory mechanism for continued circuit plasticity, synapse formation, and integration of new neurons in the adult brain.

Somatic Activating KRAS Mutations in Arteriovenous Malformations of the Brain

BACKGROUND Sporadic arteriovenous malformations of the brain, which are morphologically abnormal connections between arteries and veins in the brain vasculature, are a leading cause of hemorrhagic stroke in young adults and children. The genetic cause of this rare focal disorder is unknown. METHODS We analyzed tissue and blood samples from patients with arteriovenous malformations of the brain to detect somatic mutations. We performed exome DNA sequencing of tissue samples of arteriovenous malformations of the brain from 26 patients in the main study group and of paired blood samples from 17 of those patients. To confirm our findings, we performed droplet digital polymerase‐chain‐reaction (PCR) analysis of tissue samples from 39 patients in the main study group (21 with matching blood samples) and from 33 patients in an independent validation group. We interrogated the downstream signaling pathways, changes in gene expression, and cellular phenotype that were induced by activating KRAS mutations, which we had discovered in tissue samples. RESULTS We detected somatic activating KRAS mutations in tissue samples from 45 of the 72 patients and in none of the 21 paired blood samples. In endothelial cell–enriched cultures derived from arteriovenous malformations of the brain, we detected KRAS mutations and observed that expression of mutant KRAS (KRASG12V) in endothelial cells in vitro induced increased ERK (extracellular signal‐regulated kinase) activity, increased expression of genes related to angiogenesis and Notch signaling, and enhanced migratory behavior. These processes were reversed by inhibition of MAPK (mitogen‐activated protein kinase)–ERK signaling. CONCLUSIONS We identified activating KRAS mutations in the majority of tissue samples of arteriovenous malformations of the brain that we analyzed. We propose that these malformations develop as a result of KRAS‐induced activation of the MAPK–ERK signaling pathway in brain endothelial cells. (Funded by the Swiss Cancer League and others.)